High frequencies of polyfunctional HIV-specific T cells are associated with preservation of mucosal CD4 T cells in bronchoalveolar lavage

Abstract

The mechanisms underlying the massive gastrointestinal tract CD4 T-cell depletion in human immunodeficiency virus (HIV) infection are not well understood nor is it clear whether similar depletion is manifest at other mucosal surfaces. Studies of T-cell and virus dynamics in different anatomical sites have begun to illuminate the pathogenesis of HIV-associated disease. Here, we studied depletion and HIV infection frequencies of CD4 T cells from the gastrointestinal tract, bronchoalveolar lavage (BAL), and blood with the frequencies and functional profiles of HIV-specific T cells in these anatomically distinct sites in HIV-infected individuals. The major findings to emerge were as follows: (i) depletion of gastrointestinal CD4 T cells is associated with high frequencies of infected CD4 T cells; (ii) HIV-specific T cells are present at low frequencies in the gastrointestinal tract compared to blood; (iii) BAL CD4 T cells are not massively depleted during the chronic phase; (iv) infection frequencies of BAL CD4 T cells are similar to those in blood; (v) significantly higher frequencies and increased functionality of HIV-specific T cells were observed in BAL compared to blood. Taken together, these data suggest mechanisms for mucosal CD4 T-cell depletion and interventions that might circumvent global depletion of mucosal CD4 T cells.

Figure 1

Depletion of CD4 T cells from blood, GI tract, and BAL. (a, b) Percentage of T cells that express CD4 was measured after lymphocyte and CD3 gating in blood, GI tract, and BAL from individuals with chronic HIV infection or AIDS. (c) Percentage of blood memory CD4 T cells that express CCR5 was measured in uninfected, chronically HIV-infected individuals, and individuals with AIDS. (d) Percentage of GI tract memory CD4 T cells that express CCR5 was measured in uninfected, chronically HIV-infected individuals, and individuals with AIDS. (e) Percentage of BAL memory CD4 T cells that express CCR5 was measured in uninfected, chronically HIV-infected individuals, and individuals with AIDS. P-values represent the results of Wilcoxon’s matched pairs test (a, b) or Mann–Whitney U-test (c, d). BAL, bronchoalveolar lavage; GI, gastrointestinal.

Figure 2

HIV infection frequency of CD4 T cells and alveolar macrophages in blood, GI tract, and BAL. (a) Percentage of HIV-infected blood central memory CD4 T cells and gut memory CD4 T cells determined by qPCR for HIV gag DNA. (b) Percentage of HIV-infected blood central memory CD4 T cells, BAL memory CD4 T cells, and BAL macrophages. (c) The ratio of CCR5 mRNA in BAL memory CD4 T cells compared to BAL macrophages in three individuals. Points depicting values for the same individual are linked by a line. BAL, bronchoalveolar lavage; GI, gastrointestinal.

Figure 3

Flow cytometric analysis of cytokine production after antigenic stimulation. GI tract lymphocytes from subject 1464 were stimulated with SEB in the presence of brefeldin A for 18 h and then stained for surface and intracellular proteins as described in Methods. Lymphocytes are gated based on characteristic light-scatter properties, single lymphocytes are gated based on forward scatter height vs. forward scatter area, and live T cells are gated based on expression of CD3 without staining for the dead cell dye vivid. CD4 or CD8 T cells are then gated and memory CD4 or CD8 T cells are gated based on the characteristic expression patterns of CD27 and CD45RO. The percentage of memory CD4 and CD8 T cells that produce IFN-γ, TNF, and IL-2 can then be determined. GI, gastrointestinal.

Figure 4

HIV-specific CD8 T cells in blood and GI tract. (a) HIV-specific CD8 T-cell responses in blood and gut defined functionally. (b) HIV-specific CD8 T cells in blood and gut defined physically. GI tract and peripheral blood lymphocytes from subjects 1458, 1479, and 1456 were stimulated with overlapping HIV peptides and stained as described in Methods. The responses are shown as the percentage of memory T cells that are HIV-specific (a). Alternatively, tetramer analysis was performed on GI tract and peripheral blood lymphocytes from subjects 1430, 1458, and 1469, and the frequencies of memory CD8 T cells that bind HIV tetramers are shown (b). GI, gastrointestinal.

Figure 5

Frequency and functionality of HIV-specific T cells in blood, GI tract, and BAL. (a) HIV-specific CD8 T-cell responses in blood and gut. (b) HIV-specific CD4 T-cell responses in blood and gut. (c) HIV-specific CD8 T-cell responses in blood and BAL. (d) HIV-specific CD4 T-cell responses in blood and BAL. (e) Functionality of HIV-specific CD8 and (f) CD4 T cells. Lymphocytes were stimulated with overlapping HIV peptides and stained as described in Methods. The responses are shown as the percentage of memory T cells that are HIV specific. Circles or squares denote individuals’ responses, with some individuals responding to multiple peptide pools as denoted with multiple like-colored symbols. In addition, tetramer analysis was performed on several individuals, and the frequencies of memory CD8 T cells that bind tetramer are denoted by triangles. Points depicting values for the same individual are linked by a line. P-values represent the results of Wilcoxon’s matched pairs test. The frequency of the total HIV-specific responses producing one, two, or three cytokines was determined using SPICE as described in Methods. BAL, bronchoalveolar lavage; GI, gastrointestinal.

Figure 6

MIP-1α and MIP-1β levels in BAL and plasma. BAL fluid and cells were collected during lavage as described in Methods. Acellular BAL fluid was concentrated 4× and stored frozen until chemokine analysis. Measurements of chemokines in BAL fluid were adjusted for dilution by the urea method. Chemokines were measured by cytokine bead array on a FACSCalibur. Points depicting values for the same individual are linked by a line. BAL, bronchoalveolar lavage.

Figure 7

MIP-1β production by HIV-specific CD8 T cells in BAL. BAL lymphocytes were stimulated with overlapping HIV peptides and stained as described in Methods with the exception that anti-MIP-1β PE was substituted for anti-CD27 PE. Gated live CD8 T cells are shown and all IFN-γ also express MIP-1β. PE, phycoerythrin.